CN114948819B - Honeysuckle flower fermented product with repairing effect and preparation method thereof - Google Patents
Honeysuckle flower fermented product with repairing effect and preparation method thereof Download PDFInfo
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Abstract
The invention provides a preparation method of honeysuckle flower fermented product with restoration effect, which comprises the following steps: s1) mixing dry honeysuckle powder with enzyme solution, and performing enzymolysis to obtain honeysuckle enzymolysis product; s2) mixing the honeysuckle enzymolysis products, sterilizing, and then adding saccharomycetes and/or lactobacillus for fermentation to obtain the honeysuckle fermentation product. Compared with the prior art, the invention utilizes saccharomycetes and/or lactobacillus to ferment the honeysuckle enzymolysis product, and utilizes the microbial fermentation technology to avoid the influence of physical and chemical extraction means on active ingredients, so that the obtained honeysuckle fermentation product has high active ingredient content, higher free radical scavenging capability and repairing effect, and can be used for skin damage resistance and repairing.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a honeysuckle flower ferment with a repairing effect and a preparation method thereof.
Background
Because of its heat-clearing and detoxicating properties, honeysuckle has been widely used in traditional Chinese medicine for thousands of years. In clinical practice, more than 500 prescriptions including honeysuckle have been used to treat various diseases. Pharmacological studies show that honeysuckle has various functions of anti-inflammatory, antiviral, antidiabetic, antiallergic, antioxidant and the like, and can be used for treating various viral diseases such as SARS, H7N9 virus and infection. In addition, it is used as a health food, a cosmetic, a soft drink and an ornamental plant. Thus, the widespread market of honeysuckle has attracted a great deal of attention from many scholars who have isolated many chemical components from honeysuckle, such as organic acids, flavones, iridoids, triterpenes and volatile oils, which have been reported as active components with certain potential pharmacological actions. More and more experiments show that the honeysuckle extract can inhibit various inflammatory reactions and various inflammatory factors, and the honeysuckle is widely considered as a safe and mild anti-inflammatory medicament for treating various inflammatory diseases.
At present, the extraction of the effective components in the honeysuckle mainly comprises water extraction, alcohol reflux extraction technology, ultrasonic extraction technology, a desorption-thermal extraction two-step method and the like. The honeysuckle flower water extract, the methanol and the 70% ethanol extract have better antioxidant activity, and in-vitro DPPH scavenging activity and superoxide anion scavenging activity researches show that the flower bud water extract, the ethanol extract and the supercritical fluid extract have antioxidant effect. The comparison research on the total phenol content, the total flavone content and the antioxidant activity of the water extract, petroleum ether, ethyl acetate and n-butyl alcohol shows that the total phenol content and the total flavone content of the ethyl acetate are highest, the DPPH free radical scavenging activity and the reducing capability in vitro are optimal, the ABTS free radical and superoxide anion radical scavenging capability of the n-butyl alcohol is best, and the antioxidant activity of the water extract, the ethyl acetate and the n-butyl alcohol extract is stronger than that of the positive control butylhydroxytoluene.
Compared with chemical and physical modes, the extraction of active ingredients by using the microbial fermentation technology has the following advantages: the activity of unique and complete original natural compounds and derivatives thereof can be maintained; the biological activity and the biological adaptability can be increased, the solubility and the digestibility can be improved through fermentation, and the novel functional bioactive components can be developed; improving the flavor and natural color of fruits, vegetables and herbs; in addition, the low temperature process protects the heat sensitive phytochemicals. Therefore, the development of the honeysuckle flower ferment with the repairing effect has important practical significance.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a honeysuckle flower ferment with repairing effect and a preparation method thereof.
The invention provides a preparation method of honeysuckle flower fermented product with restoration effect, which comprises the following steps:
s1) mixing dry honeysuckle powder with enzyme solution, and performing enzymolysis to obtain honeysuckle enzymolysis product;
s2) sterilizing the honeysuckle enzymolysis product, and then adding saccharomycetes and/or lactobacillus for fermentation to obtain the honeysuckle fermentation product.
Preferably, the enzyme solution comprises cellulase and pectinase; the mass ratio of the cellulase to the pectase is 1: (0.5-2).
Preferably, the enzyme activity of the cellulase in the enzyme solution is 2500-3500U/mL; the enzyme activity of pectase in the enzyme liquid is 2000-3000U/mL;
the mass ratio of the enzyme liquid to the honeysuckle flower dry powder is (15-25): 1.
preferably, the enzymolysis temperature is 40-60 ℃; the enzymolysis time is 2-4 h.
Preferably, the step S1) is carried out enzymolysis and then centrifugation to obtain honeysuckle flower enzymatic hydrolysate; the rotational speed of the centrifugation is 6000-12000 r/nmin; the centrifugation time is 10-20 min.
Preferably, the number of viable bacteria of the yeast is 1×10 or more 7 CFU/mL; the viable count of the lactobacillus is more than or equal to 1 multiplied by 10 8 CFU/mLThe method comprises the steps of carrying out a first treatment on the surface of the The mass of the saccharomycete is 3% -7% of the mass of the honeysuckle enzymolysis product; the mass of the lactobacillus is 5-15% of the mass of the honeysuckle enzymolysis product.
Preferably, the saccharomycete is lactobacillus plantarum; the saccharomycete is saccharomyces cerevisiae.
Preferably, the lactic acid bacteria comprise lactobacillus plantarum cctccc No: m20191045 and/or lactobacillus plantarum ACCC 03954;
the yeasts include Angel wine yeast SY and/or Saccharomyces cerevisiae ACCC 21188.
Preferably, the fermentation temperature is 28-37 ℃; the fermentation time is 2-4 days.
The invention also provides the honeysuckle flower fermented product with the repairing effect prepared by the preparation method.
The invention provides a preparation method of honeysuckle flower fermented product with restoration effect, which comprises the following steps: s1) mixing dry honeysuckle powder with enzyme solution, and performing enzymolysis to obtain honeysuckle enzymolysis product; s2) mixing the honeysuckle enzymolysis products, sterilizing, and then adding saccharomycetes and/or lactobacillus for fermentation to obtain the honeysuckle fermentation product. Compared with the prior art, the invention utilizes saccharomycetes and/or lactobacillus to ferment the honeysuckle enzymolysis product, and utilizes the microbial fermentation technology to avoid the influence of physical and chemical extraction means on active ingredients, so that the obtained honeysuckle fermentation product has high active ingredient content, higher free radical scavenging capability and repairing effect, and can be used for skin damage resistance and repairing.
Drawings
FIG. 1 is a bar graph of the effect of a sample on the fruit acid damage of keratinocytes in an embodiment of the present invention;
FIG. 2 is a micrograph of the effect of a sample on keratinocyte fruit acid damage in an example of the present invention;
FIG. 3 is a bar graph showing the effect of samples on SLS damage to keratinocytes in an example of the present invention.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention provides a preparation method of honeysuckle flower fermented product with restoration effect, which comprises the following steps: s1) mixing dry honeysuckle powder with enzyme solution, and performing enzymolysis to obtain honeysuckle enzymolysis product; s2) mixing the honeysuckle enzymolysis products, sterilizing, and then adding saccharomycetes and/or lactobacillus for fermentation to obtain the honeysuckle fermentation product.
The source of all the raw materials is not particularly limited, and the raw materials are commercially available.
Mixing the honeysuckle dry powder with enzyme solution for enzymolysis; the honeysuckle flower dry powder is the dry powder of honeysuckle flower of the family Caprifoliaceae, which is well known to the person skilled in the art, and is not particularly limited, and the honeysuckle flower dry powder is preferred in the invention; the enzyme solution preferably comprises cellulase and pectase; the mass ratio of the cellulase to the pectase is preferably 1: (0.5 to 2), more preferably 1: (0.8 to 1.5), and more preferably 1: (0.8 to 1.2), most preferably 1:1, a step of; the enzyme activity of the cellulase in the enzyme liquid is preferably 2500-3500U/mL, more preferably 2800-3200U/mL, and even more preferably 3000U/mL; the enzyme activity of the pectase in the enzyme liquid is preferably 2000-3000U/mL, more preferably 2200-2800U/mL, even more preferably 2400-2600U/mL, and most preferably 2500U/mL; the mass ratio of the enzyme solution to the honeysuckle flower dry powder is preferably (15-25): 1, more preferably (18 to 22): 1, more preferably 20:1, a step of; the enzymolysis temperature is preferably 40-60 ℃, more preferably 45-55 ℃ and still more preferably 50 ℃; the time for the enzymolysis is preferably 2 to 4 hours, more preferably 1.5 to 3.5 hours, still more preferably 3 hours.
Preferably centrifuging after enzymolysis to obtain supernatant, namely the honeysuckle flower hydrolysate; the rotation speed of the centrifugation is preferably 6000-12000 r/nmin, more preferably 8000-10000 r/nmin, and still more preferably 10000r/nmin; the time of the centrifugation is preferably 10 to 20 minutes, more preferably 15 minutes.
Mixing the honeysuckle enzymolysis products for sterilization; preferably, the honeysuckle flower enzymatic hydrolysate is mixed; the temperature of the sterilization treatment is preferably 110-120 ℃, more preferably 115 ℃; the time for the sterilization treatment is preferably 15 to 30 minutes, more preferably 15 to 25 minutes, and still more preferably 20 minutes.
Then adding saccharomycetes and/or lactobacillus for fermentation; the mass of the saccharomycete is preferably 3-7% of the mass of the honeysuckle enzymolysis product, and more preferably 5%; the saccharomycete is preferably saccharomyces cerevisiae, more preferably Angel wine yeast SY and/or saccharomyces cerevisiae ACCC 21188; the viable count of the yeast is preferably 1×10 or more 7 CFU/mL; in the invention, the saccharomycete is preferably obtained by activating the following steps: inoculating a saccharomycete strain into a YPD culture medium, and obtaining saccharomycete through culture passage; the temperature of the culture is preferably 25℃to 35℃and more preferably 30 ℃; the time of the culture is preferably 36-48 hours; the number of passages is preferably 3; the mass of the lactobacillus is preferably 5-15% of the total mass of the honeysuckle enzymolysis product, more preferably 8-12%, and still more preferably 10%; the lactobacillus is preferably lactobacillus plantarum, more preferably with a preservation number of CCTCC No: lactobacillus plantarum Picp-2 of M20191045 and/or lactobacillus plantarum ACCC 03954; the viable count of the lactic acid bacteria is preferably 1×10 or more 8 CFU/mL; the lactobacillus is preferably obtained by activating the following steps: inoculating lactobacillus strains into an MRS culture medium, and culturing and passaging to obtain lactobacillus; the temperature of the culture is preferably 25℃to 35℃and more preferably 30 ℃; the time of the culture is preferably 24 hours or more; the number of passages is preferably 3. The fermentation temperature is preferably 28-37 ℃; in the present invention, when fermentation is performed using only lactic acid bacteria, the temperature of fermentation is preferably 37 ℃; when the fermentation system contains saccharomycetes, the temperature of fermentation is preferably 28 ℃; the fermentation time is preferably 2 to 4 days, more preferably 3 days.
After fermentation, preferably centrifuging, and collecting supernatant to obtain a honeysuckle flower fermented product; the rotation speed of the centrifugation is preferably 6000-12000 r/nmin, more preferably 8000-10000 r/nmin, and still more preferably 10000r/nmin; the time of the centrifugation is preferably 10 to 20 minutes, more preferably 15 minutes.
The invention utilizes saccharomycetes and/or lactobacillus to ferment the honeysuckle enzymolysis product, and utilizes the microbial fermentation technology to avoid the influence of physical and chemical extraction means on active ingredients, so that the obtained honeysuckle fermentation product has high active ingredient content, higher free radical scavenging capability and repairing effect, and can be used for skin damage resistance repair.
The invention also provides the honeysuckle flower fermented product with the repairing effect, which is prepared by the preparation method.
In order to further illustrate the invention, the following examples are combined to describe the honeysuckle flower ferment with repairing effect and the preparation method thereof in detail.
The reagents used in the examples below are all commercially available; lactobacillus plantarum ACCC03954 and saccharomyces cerevisiae ACCC21188 used in the examples were purchased from the chinese agricultural microorganism strain collection management center; hacat human immortalized keratinocytes (Bio-51586) used in the examples were purchased from the microorganism strain query net (https:// www.biobw.org /).
Comparative example 1
1.1 Water extraction of mountain silver flower
According to pure water: mixing flos Lonicerae dry powder=20:1, extracting at 90deg.C for 3 hr, centrifuging at 9000r/min for 15min, and collecting supernatant for index detection to obtain flos Lonicerae water extract.
1.2 preparation of post-water extraction ferment of lonicera confusa:
a) Fermentation strain selection
Lactic acid bacteria: lactobacillus plantarum (lactobacillus plantarum Picp-2 with a preservation number of CCTCC No. M20191045) and lactobacillus plantarum ACCC 03954;
yeast: angel wine Yeast SY and Saccharomyces cerevisiae ACCC 21188.
b) Fermentation strain activation
Yeast: inoculating the strain into YPD culture medium, culturing at 30deg.C for 36-48 hr, and continuously passaging for 3 times to fully activate the strain until the number of viable bacteria is 1.0X10 7 And obtaining the yeast SY by CFU/mL.
Lactic acid bacteria: will beInoculating strain into MRS culture medium, culturing at 35deg.C for 24 hr, and continuously passaging for 3 times to fully activate strain until viable count is 1.0X10 8 And (3) obtaining the lactic acid bacteria Picp-2 by CFU/mL.
c) Post-extraction fermentation of flos Lonicerae
Lactic acid bacteria and saccharomycetes are mixed and fermented: 200mL of the aqueous extract of flos Lonicerae obtained in 1.1, sterilizing at 115deg.C for 20min, adding 10% lactic acid bacteria Picp-2+5% Angel wine yeast SY or (Lactobacillus plantarum ACCC 03954+Saccharomyces cerevisiae ACCC 21188), and fermenting at 28deg.C for 3 days. Centrifuging, collecting supernatant to obtain the flos Lonicerae fermentation product after water extraction, and centrifuging for 15min at 9000r/min to obtain flos Lonicerae fermentation product for index detection.
Example 1
1.1 enzymatic hydrolysis of honeysuckle
Crude enzyme liquid (pectase and cellulase 1:1; pectase enzyme activity: 2500U/mL; cellulase enzyme activity: 3000U/mL): mixing flos Lonicerae dry powder=20:1, extracting at 50deg.C for 3 hr, centrifuging at 9000r/min for 15min, and collecting supernatant for index detection to obtain flos Lonicerae enzymolysis product.
1.2 preparation of post-enzymatic fermentation product of lonicera confusa
Fermentation strain selection and activation
a) Fermentation strain selection
Lactic acid bacteria: lactobacillus plantarum (lactobacillus plantarum Picp-2 with a preservation number of CCTCC No. M20191045) and lactobacillus plantarum ACCC 03954;
yeast: angel wine Yeast SY and Saccharomyces cerevisiae ACCC 21188.
b) Fermentation strain activation
Yeast: inoculating the strain into YPD culture medium, culturing at 30deg.C for 36-48 hr, and continuously passaging for 3 times to fully activate the strain until the number of viable bacteria is 1.0X10 7 CFU/mL or more.
Lactic acid bacteria: inoculating strain into MRS culture medium, culturing at 35deg.C for 24 hr, and continuously passaging for 3 times to fully activate strain until viable count is 1.0X10 8 CFU/mL or more.
c) Post-enzymolysis fermentation of lonicera japonica
Fermenting by using saccharomycetes alone: 200mL of the flos Lonicerae enzymolysis product obtained in 1.1, sterilizing at 115deg.C for 20min, adding 5% yeast SY or Saccharomyces cerevisiae ACCC21188, and fermenting at 28deg.C for 3 days.
Fermentation of lactic acid bacteria alone: 200mL of the honeysuckle enzymolysis product obtained in 1.1, sterilizing at 115 ℃ for 20min, adding 10% of lactobacillus Picp-2 or lactobacillus plantarum ACCC03954, and fermenting at 37 ℃ for 3 days.
Lactic acid bacteria and saccharomycetes are mixed and fermented: 200mL of the flos Lonicerae zymolyte obtained in 1.1, sterilizing at 115deg.C for 20min, adding 10% lactic acid bacteria Picp-2+5% yeast SY or (ACCC 03954+ACCC21188), and fermenting at 28deg.C for 3 days. Centrifuging, collecting supernatant to obtain the fermentation product after enzymolysis of the lonicera japonica, and centrifuging for 15min at 9000r/min to obtain the fermentation product for index detection.
1.3 index detection
(1) Measurement of DPPH radical scavenging ability:
the experimental group was added with 1ml of sample solution (fermentation product of lonicera japonica) +1ml of 0.2mmol/L DPPH ethanol solution. Control group was added with 1ml absolute ethanol+1 ml stock solution. The blank was added with 1ml of absolute ethanol+1 ml of a 0.2mmol/L solution of DPPH ethanol, and the mixture was allowed to stand at room temperature (25 ℃) for 30 minutes. 517 and n m, 300. Mu.L was aspirated, and the absorbance of the test group was measured and recorded as A i . The absorbance of the control group was measured and designated A j . Blank set is denoted as A0. Each sample was measured 3 times in parallel.
DPPH clearance (%) = [1- (a) i -A j )/A 0 ]×100％
(2) Superoxide anion radical scavenging ability assay:
adding 5.7mL Tris-HCl buffer solution (50 mmol/L, pH=8.2) into a 10mL test tube, adding 0.2mL sample solution (flos Lonicerae ferment) for mixing, placing into a 25 ℃ incubator, taking out after 10min, adding 0.1mL (10 mmol/L) o-triphenyl aqueous solution (preheated), rapidly mixing, measuring the increase value (Aj) of absorbance within 1min at 320nm wavelength by a multifunctional enzyme-labeling instrument, calculating the increase value of absorbance per 1min in a linear range, taking the above reagent, replacing the sample solution with equal volume of water, measuring the increase value (A i )。
Superoxide anion radical scavengingRate (%) = (a i -A j )/A i ×100％
(3) Determination of the scavenging ability of hydroxyl radicals (. OH):
hydroxyl radical (. OH) scavenging test method referring to Fenton reaction method, sequentially adding ferrous sulfate solution (6 mmol/L), hydrogen peroxide solution (6 mmol/L) and sample solution (flos Lonicerae fermented product) into test tube, standing for 10min after 2mL each, adding 2mL salicylic acid solution (6 mmol/L), standing for 30min, and measuring absorbance A at 510nm 0 The same treatment as that of distilled water was used instead of the sample solution to determine the absorbance A x 。
Hydroxyl radical (·oh) clearance (%) = (1-a) 0 /A x )×100％
(4) Determination of ABTS clearance:
0.2mL 7.4mmoL/L ABTS and 0.2mL 2.6mmoL/L K 2 S 2 O 8 Mixing, reacting for 12h at room temperature in dark place, diluting with 95% ethanol (pH=7.4 phosphate buffer solution, 95% ethanol or methanol) for 40-50 times after the reaction is completed, so that the absorption value of the mixed solution is 0.68-0.72 under 734nm luminosity, and obtaining the working solution.
Diluting the fermentation sample solutions (flos Lonicerae fermented product) with different treatments, respectively sucking 0.2mL, adding 0.8mL of diluted working solution, mixing, standing for reaction for 6min, and rapidly measuring absorbance A at 734nm wavelength 0 . The absorbance A was measured by the same treatment as that using 95% ethanol instead of the sample solution x . Each sample was measured 3 times in parallel.
ABTS radical clearance (%) =1-a 0 /A x ×100％
(5) Total reducing force detection
Transferring 1ml of sample solution (flos Lonicerae ferment) into 10ml of EP tube, immediately rapidly adding 2.5ml (0.2 ml/L) PBS solution and 2.5ml 1% potassium ferricyanide solution, mixing, keeping temperature in water bath at 50deg.C for 20min, taking out, adding 2.5ml 10% trichloroacetic acid solution, adding trichloroacetic acid, centrifuging at 3500r/min for 10min at room temperature if precipitation occurs, absorbing 2.5ml supernatant, and adding 2.5ml distilled water; if no precipitate is formed after the addition of trichloroacetic acid, 2.5ml of distilled water is added after sucking up 2.5ml of the solution. 0.5ml of 0.1% ferric trichloride solution was added thereto, and the mixture was uniformly mixed and reacted for 10 minutes. The control group uses distilled water with equal amount to replace the sample liquid, and other operations are unchanged. And finally, measuring the absorbance value under 700nm luminosity, wherein the absorbance value of the experimental group and the control group is in direct proportion to the reducing power.
(6) Determination of total polyphenol content
1mg/mL gallic acid standard solution 0, 20, 40, 60, 80, 100, 200, 400 and 600. Mu.L was accurately removed under room temperature conditions, placed in a test tube and made up to 1mL with pure water. After 1.5mL of Folin-Ciocalteu reagent solution was added to react for 3 to 8 minutes, 1mL of 20% (g/v) Na was added 2 CO 3 The solution was mixed well and the volume was set to 10mL with double distilled water. After 60min of reaction at room temperature, absorbance at 760nm was measured, and total polyphenol content (x) was represented by gallic acid (mg/mL) and absorbance at 760nm (y) was represented by ordinate. 100. Mu.L of sample liquid (the fermentation product of the lonicera japonica) is sucked, the absorbance value is measured according to Fu Lin Fenfa, and then the total polyphenol content is calculated according to a standard curve.
(7) Determination of total flavone content
Drawing and process of rutin standard curve
Preparing standard solution, namely placing 5mg of rutin standard into a 25ml volumetric flask, adding 70% ethanol until the volume is equal to the volume, shaking uniformly to obtain 0.2mg/ml rutin standard solution, and respectively weighing 0.00, 1.00, 2.00, 3.00, 4.00, 5.00 and 6.00ml rutin standard solution, and respectively placing into the 25ml volumetric flask.
Adding 5.00ml of water and 1.00ml of 5% sodium nitrite solution, shaking uniformly, and standing for 6min;
adding 1.00ml of 10.00% aluminum nitrate solution, shaking uniformly, and standing for 6min;
10.00ml of 4.00% sodium hydroxide solution is added, water is added to the scale respectively, and the mixture is kept stand for 15min;
in addition, a solution without standard was used as a blank.
Scanning with ultraviolet spectrophotometry Ji Quanbo with a length of 200-800 nm to obtain a maximum absorption wavelength of 510nm, measuring absorbance at the wavelength of 510nm, and preparing a standard curve by taking the concentration C as an abscissa and the absorbance A as an ordinate.
Determination of total flavone content in sample solution
1ml of the sample solution to be detected (honeysuckle flower ferment) is taken, kept stand for 15 minutes according to a rutin standard curve drawing method, absorbance A is measured at the wavelength of 510nm, and the content (mg/ml) of total flavonoids in the solution to be detected is calculated according to a regression equation.
1.4 cell protocol
The damage model of human skin keratinocyte is established through the induction of the fruit acid and SLS damage, and the damage protection effect of the object to be detected is detected through an MTT colorimetric method, so that experimental basis is provided for the product development of the object to be detected in the field of damage resistance repair.
(1) Cell plating: the cell suspension density was adjusted to 0.8X10 5 Inoculating into 96-well plate at a volume of 100 μl/well, simultaneously arranging cell negative hole, damage positive hole and zeroing hole, placing at 37deg.C, 5% CO 2 The cells were cultured in an incubator for 24 hours.
(2) Fruit acid or SLS induced oxidative damage: discarding the old culture medium, diluting the fruit acid or SLS mother liquor to 0.3% with the culture medium, adding cells, 100 μl/well, adding the complete culture medium into cell negative control well and zeroing well, placing at 37deg.C, 5% CO 2 Culturing in a cell culture box for 2-3 h and discarding.
(3) Diluting and sample adding of the object to be detected: diluting the test substance to test concentration by using complete culture medium as diluent, adding 100 μl/well of test substance to cells, adding equivalent complete culture medium to cell control well, damage positive well and zeroing well, placing at 37deg.C, 5% CO 2 Culturing in a cell culture box for 24 hours.
(4) MTT detection: the test was carried out according to the method of (4).
(5) And (3) calculating: the method of calculation as in (5).
(6) Statistical analysis: and 1..6.
1.5 experimental results
TABLE 1 index content of fermented product
(7) Results of the efficacy test for repair of fruit acid lesions are shown in fig. 1 and 2. In FIG. 1, if the cell viability of the sample is higher than that of the NC group, it is shown that it has a reparative effect; otherwise, the method has no effect. In fig. 2, BC is a blank control group and NC is a model lesion group. As can be seen from fig. 1 and fig. 2, in the normal state, the BC blank control group cells are normally attached, the cells are in a stone paving shape, the NC group cells are abnormal in morphology and irregular in edge after being treated by fruit acid, and part of the cells fall off or even die, so that the cell viability of the NC group is reduced to about 43% compared with that of the BC group; the SY+Picp-2(1), SY+Picp-2(2) and SY+Picp-2(3) lonicera japonica fermentation liquid with the test concentration of 1%, 0.1% and 0.01% has obvious repairing effect on the keratinocytes damaged by the fruit acid by combining the quantitative and cytomorphological observation results.
(8) The results of the SLS injury repair efficacy test are shown in fig. 3. In FIG. 3, if the cell viability of the sample is higher than that of the NC group, it is indicated that it has a reparative effect, and vice versa. BC is the blank control group and NC is the model lesion group. As can be seen from fig. 3, the NC group cell morphology was significantly changed after SLS treatment, the cell contraction morphology was abnormal, and a part of the cell gap was enlarged, dropped and even killed, and the NC group cell viability was reduced to about 75% compared with the BC group. The test concentration is 0.01% SY+Picp-2(1), 0.01% SY+Picp-2(2), 0.1% SY- (2), 0.1% enzymolysis, 1% non-enzymolysis, 0.1% and 0.01% lonicera japonica fermentation liquid have a certain repairing effect on the SLS injury of the keratinocytes. The lonicera confusa fermentation liquor with other test concentration has no obvious effect of repairing keratinocyte damaged by SLS.
Claims (5)
1. The preparation method of the honeysuckle flower ferment with the repairing effect is characterized by comprising the following steps of:
s1) mixing dry honeysuckle powder with enzyme solution, and performing enzymolysis to obtain honeysuckle enzymolysis product;
s2) sterilizing the honeysuckle enzymolysis product, and then adding saccharomycetes and lactobacillus for fermentation to obtain a honeysuckle fermentation product;
the enzyme solution comprises cellulase and pectase; the mass ratio of the cellulase to the pectase is 1: (0.5-2);
the enzyme activity of the cellulase in the enzyme liquid is 2500-3500U/mL; the enzyme activity of pectase in the enzyme liquid is 2000-3000U/mL;
the mass ratio of the enzyme liquid to the honeysuckle flower dry powder is (15-25): 1, a step of;
the number of viable bacteria of the yeast is more than or equal to 1 multiplied by 10 7 CFU/mL; the viable count of the lactobacillus is more than or equal to 1 multiplied by 10 8 CFU/mL; the mass of the saccharomycete is 3% -7% of the mass of the honeysuckle enzymolysis product; the mass of the lactobacillus is 5% -15% of the mass of the honeysuckle enzymolysis product;
the lactobacillus comprises lactobacillus plantarum CCTCC No: m20191045 and/or lactobacillus plantarum ACCC 03954;
the yeasts include Angel wine yeast SY and/or Saccharomyces cerevisiae ACCC 21188.
2. The method according to claim 1, wherein the temperature of the enzymolysis is 40 ℃ to 60 ℃; the enzymolysis time is 2-4 h.
3. The preparation method according to claim 1, wherein the step S1) is performed with enzymolysis and centrifugation to obtain honeysuckle enzymolysis product; the rotational speed of the centrifugation is 6000-12000 r/nmin; the centrifugation time is 10-20 min.
4. The method according to claim 1, wherein the fermentation temperature is 28 ℃ to 37 ℃; the fermentation time is 2-4 days.
5. The lonicera japonica fermentation product with repairing effect prepared by the preparation method of any one of claims 1 to 4.
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| CN108420758A (en) * | 2018-03-30 | 2018-08-21 | 深圳市芭格美生物科技有限公司 | A kind of biological enzyme processing and corrosion-resistant honeysuckle flowers plant extraction liquid and preparation method thereof |
| CN111973647A (en) * | 2019-05-21 | 2020-11-24 | 大江生医股份有限公司 | Preparation method of honeysuckle flower fermentation product and application of honeysuckle flower fermentation product in improving skin appearance and resisting aging |
| CN114009754A (en) * | 2021-08-31 | 2022-02-08 | 陕西佰瑞衡健康科技有限公司 | Preparation method of fermented plant exosomes |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108420758A (en) * | 2018-03-30 | 2018-08-21 | 深圳市芭格美生物科技有限公司 | A kind of biological enzyme processing and corrosion-resistant honeysuckle flowers plant extraction liquid and preparation method thereof |
| CN111973647A (en) * | 2019-05-21 | 2020-11-24 | 大江生医股份有限公司 | Preparation method of honeysuckle flower fermentation product and application of honeysuckle flower fermentation product in improving skin appearance and resisting aging |
| CN114009754A (en) * | 2021-08-31 | 2022-02-08 | 陕西佰瑞衡健康科技有限公司 | Preparation method of fermented plant exosomes |
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| Title |
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| 酿酒酵母和植物乳杆菌混合发酵对金银花浸提液多酚物质的影响;崔亚鹏等;《食品与发酵工业》;第48卷(第5期);第95页左栏第1段，第96页第1.3.2节，第98页右栏2段 * |
| 金银花绿原酸酶法提取新工艺研究;刘佳佳等;《中成药》;第24卷(第6期);第416页摘要，第417-418页第2.4节 * |
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